Sticky proteins, complexity drama and selection's blind eye

*For your entertainment, rejected titles:
[Sticky proteins and complex relationships]
[(protein) Relationship drama: promiscuous proteins in small populations]
[Not all is good that sticks: non-adaptive complexity gain through compensatory protein adhesion]
[Man, I suck at titles]

NB: This post can be considered as part 2.5 of my In defense of constructive neutral evolution series; also recommended for some background are part 1, discussing selection, drift and Neutral Theory, and part 2, discussing Constructive Neutral Evolution; to answer a popular question, part 3 *will* materialise eventually once I get off my ass and write it.

Constructive neutral evolution is one mechanism of complexity increase without any associated increase in fitness – or, in other words, non-adaptive complexity gain. Basically, a random interaction between two proteins can lead to a fixed dependency if this interaction compensates for a mutation that was otherwise lethal – termed 'pressuppression'. In this way, previously unnecessary dependencies accumulate to make a very bulky, bureaucratic system that essentially does the same thing. We've all seen it in our institutions, and evolution is about as efficient.

Now, one bottleneck in this model is waiting for proteins to actually interact. Proteins are quite sticky and non-specific by nature, but usually not too much as that can be quite deleterious. Piling up a bunch of proteins on each other has a non-negligible chance of interfering with their function, and one would expect for chance interactions to not be excessively promiscuous, although those who have done regulatory genetics and protein work are probably aware just how annoyingly non-specific some of the protein binding can get. Luckily, there is now a possibly mechanism boosting these chance interactions, and thus alleviating that particular bottleneck in the Constructive Neutral Evolution process, rapidly accelerating complexification and protein network obfuscation to the extent where the interaction map looks like a web; not a finely organised web of an orb-weaver but rather one of those clumpy webs that are a clusterfuck of stickiness and silk. Enter this week's Fernández and Lynch 2011 Nature paper, from here onwards referred to as "the paper".

Protein 'stickiness' can be enhanced by biochemical means. Proteins vary in stability, and themselves come in populations – generally, most are in the optimal conformation that is presumably functional, but some individuals are messed up. This happens well past the sequence and folding errors, and some perfectly 'normal' proteins can be in a suboptimal state at any given time. Clearly, this affects the overall efficiency of the protein – even if it's enzymatically awesome, the overall 'protein' as we biologists understand it (sans population aspect) would decline in efficiency if a large chunk of its population is in a misfolded state.

One aspect that pushes around the proportion of the protein in the 'right' conformation is how well it plays with water. It shouldn't be too surprising that hydrophobic regions induce instability. What was new to me, but perhaps old news to those who actually understood chemistry, is that the exposure of the polar(hydrophilic) protein backbone to water also has a destabilising effect – and not only that, but often more significant than that of exposed hydrophobic regions! This may seem counterintuitive – doesn't water like hydrophilic regions? And there lies our problem.

Water molecules are attracted to polar groups, and the amino acid backbone is quite polar. This means little water molecules wander in towards the backbone and form hydrogen bonds with it. The problem is twofold: first of all, the protein, like all molecules, likes to 'jiggle'. The more it can jiggle in its given conformation, the more favourable that conformation is thermodynamically since its satisfied by more states. Entropy, etc. (now we're *really* entering territory I know nothing about, since my phys chem experience is locked away by PTSD...). Hooking up this backbone with water molecules reduces its 'jiggle' room, and makes it less thermodynamically stable – making change to other conformations more probable, therefore possibly leading to more errors in the protein population.

Secondly, as detailed further in the paper, water likes to hang out with more of itself. Water molecules are happiest in foursomes, sharing four hydrogen bonds with their neighbours. When a creepy protein backbone emerges and lures an unsuspecting water molecule away into the protein's murky depths, the water molecule cannot form as many bonds with its fellows (or as many hydrogen bonds, period), and is really sad and lonely. Or, in proper terms, the system becomes less stable, since thermodynamics will favour an arrangement where these water molecules are all happily coordinated with each other, and not being molested in a corner by an amino acid polar group. In other words, exposing the polar backbone (Solvent-Accessible Backbone Hydrogen Bonds, SABHBs in the paper) to water induces what is called Protein-Water Interfacial Tension (PWIT).

One way this tension can be released and the backbone exposure ('coded for' by genes, by the way) can be compensated for is if a random other protein (or more of its own kind) are recruited to cover that exposed backbone. This would help stabilise the protein conformation, and allow this potentially deleterious drawback to be tolerated (and get fixed in the population). Ultimately, the second (and third, etc) protein can become exapted for something useful, although just an eventual dependency is good enough to make sure these proteins stick together permanently. The crazy web of interactions gets crazier.

Fernández & Lynch's fig1a suffices perfectly but I like making diagrams, so I made one anyway. See text.

(Disclaimer: I'm horrible at chemistry, this may all have been thoroughly wrong...read the paper.)

Now I'm about the last person to willingly blog about biochemistry, and this seems to have little only a distant relevance to evolution, particularly the non-adaptive kind that fascinates yours truly. It will make sense in a bit. Recall from a few seconds ago (hey, already difficult for some of us) that protein instability leads to reduced protein efficiency. This reduction is generally tolerated, however, until it's bad enough to have a higher chance of being removed. Recall from [what should be] introductory population genetics that selection acts probabilistically, with true slightly deleterious mutations have a lesser, but still significant, chance of fixation than strongly deleterious mutations, which selection has a higher chance of taking care of before drift quietly fixes it. (more detail in older post here) Since proteins are, quite unsurprisingly, also governed by fundamental principles of population genetics, drift becomes involved there too.

As populations get smaller, drift becomes a more dominant force relative to selection, and the window of 'effectively neutral' mutations – slightly beneficial and slightly deleterious, but unlikely to be dealt with by selection – increases. More mess is tolerated. This means more protein inefficiencies are allowed to fix in the population, those induced by backbone exposure among them. Since there are now more proteins that are no longer happy with themselves (or, rather, have an increased Protein-Water Interfacial Tension), they are more likely to stick together for biochemical stability. And here Constructive Neutral Evolution can come in too, allowing further deleterious mutations that are now presuppressed by the recruited proteins. In a way, this greases the presuppression process, rather than competing with it as this BBC news piece made Ford Doolittle appear to suggest.

Now, this is all great in theory, but is there any real data in support of this? For one thing, there is a clear increase of interactome (set of all interactions in an organism) complexity correlating with decrease in effective population size, suggesting a link between lax selection and accumulating complexity. Furthermore, the proteins in organisms of these smaller populations have more blistering backbone exposures to water. Supporting the relationship with population size further yet with the advantage of more phylogenetically independent events (but less interactome data), bacterial intracellular endosymbionts consistently exhibit higher protein backbone exposure (hydration) than their free-living counterparts. Selection appears to disfavour not only polar backbone exposure (also described as 'poorly wrapped proteins' in the paper), but once again, the rise of interaction complexity as a whole. (Fernández and Lynch 2011 Nature, in case you somehow managed to miss that)

Obviously I like this paper because it adds another mechanism to the arsenal of evolutionary processes happening independently of adaptation. But moreover, I don't think one can find too many examples of biochemistry mixed with population genetics. You hardly find cell and developmental biologists thinking about population genetics, and perhaps many biochemists have never even been exposed to such a subject. When fields that should never come that close together do, some really nice explosions of insight can occur (my sad attempt at chemical metaphors). We really need to talk to other more, and maybe even wander over to other departments from time to time. It's sometimes (often) frustrating to communicate with those strange ones from afar, but just like ethnic xenophobia, its interdisciplinary counterpart must also be overcome.

-----
Figure 2a annoyed me a little as it ignored phylogenetic relationships, which is a big no-no when comparing properties of taxa. The figure is technically fine, especially since there aren't any correlation analyses there, but it's hard to discount phylogenetic history as being the cause behind the correlation of the traits without actually the characters on a tree. Anyway, since I like playing with data and running statistical analyses on things, especially when I didn't actually have to go through the pain of obtaining the data myself, I mapped some characters (interactome complexity from fig2a) on a phylogeny:

Unfortunately, even the most basic statistical operations become an epic headache when trees are involved, and very quickly things become painfully complicated, for the human as well as the computer. Especially when you're handed a dataset of mixed categorical and continuous characters, as I learned the hard way last night. After fighting Mesquite for a good many hours, I finally had to resort to extracting the Ne*µ (effective pop size * mutation rate; roughly put, both lead to increased selection efficiency) estimates from Lynch & Conery 2003 – relying on an intersection of two datasets meant that our taxon sampling was quite sad by the end of this enterprise. Anyway, I ran a pairwise comparison test (Maddison 1999 J Theor Biol) on the data, which probably isn't the best thing ever, but I got something resembling significance: p = 0.019. Depending on how statistically noisy your field is, you may even deem this acceptable. In any case, not too bad given my crude (and somewhat clueless) analysis and limited taxon sampling:

Moral of the story: the inverse correlation between interactome complexity and effective population size is unlikely to be a mere artefact of shared phylogenetic history. In other words, Fernández & Lynch's hypothesis stands strong.

I mostly did this because I thought it'd take a couple hours max. If hours meant days, that wasn't too far off... but hey, I learned something!

Acknowledgments: thanks to Lucas Brouwers for helping me wade through the heavy biochemical stuff, and to Mike Lynch for explaining the key idea of the paper a while earlier. Otherwise I would've probably been too daunted to even read it, let alone blog about it...
Oh, and my Twitter people for random phylogenetics advice ;-)

Reference
Fernández, A., & Lynch, M. (2011). Non-adaptive origins of interactome complexity Nature DOI: 10.1038/nature09992

[will add some supplementary refs once I return to internet on Monday...]

Ratcheting up some splice leaders: a note on directionality

In the sea of eukaryotic genetic diversity also lurk different manners of doing day-to-day genome work itself. Ciliates run two nuclear genomes, trypanosome kinetoplasts contain a chainmail suit of RNA editing circles and dinoflagellates are just weird in every genome compartment they have. Their plastids contain tiny minicircles often containing but a single gene, capable of "rolling" transcription where the minicircle is much like a Mesopotamian cylindrical seal, leaving a concatenated repeated string of genes on the transcript. The mitochondria have linear genomes with short fragmented repeated chunks of important genes all over them. But the nuclear genome is the most fucked up: for one thing, dinoflagellates lack a few histones, and have enormous genomes stored in absolutely bizarre chromosomes. More importantly for our story: every single gene must be trans-spliced with a 'splice leader', a short sequence that attaches at the beginning of the mRNA transcript and brings to it the 3' cap necessary for transcription to work. Oddly enough, Euglenozoans like the trypanosomes and euglenids seem to have a very similar system, evolved entirely by chance* convergence (Lukes et al. 2009 PNAS goes over this remarkable convergence in more detail).

*Or perhaps something happened to both that made them prone to evolve this bizarre system.

Genomic quirks are not just interesting in their own right as some arcane oddities, but can reveal a great deal about the dynamics of genomes in general. The dinoflagellate splice leader system turns out to yield a very crisp illustration of the power of ratchets and the toll of reverse transcription on genomes.

To reiterate, every single nuclear gene transcript in a dinoflagellate must be spliced with the 3'cap-bearing 'splice leader', or else it simply won't work. This means that the dino is full of mature transcripts with splice leaders attached to the transcribed genes. Enter reverse transcriptases, which are prevalent in probably most, if not all, eukaryotic genomes, thanks to viruses and their partners in genomic parasitism crimes, transposons. When they're not busy moving transposons around and helping viruses move in, they reverse transcribe random gene transcripts for fun, that may then, on occasion, be successfully recombined back into the genome. This process probably doesn't happen [successfully] every day, but over thousands or millions of years (and countless individuals) is rampant enough to leave a noticeable trace in the genome.

So we have a load of transcripts floating around with an extra sequence stitched onto them from the splice leader. Do the reverse transcriptases care in the slightest? Of course not: to them, a ribonucleotide is a ribonucleotide, give or take some trace biophysical stuff that might make a couple people cringe at what I just said. (meaning, I wouldn't be surprised if there could be some slight but ultimately detectable biases there too) This means that splice leader, on occasion, actually makes its way back into the nuclear genome attached to the beginning of the gene.

However, this splice leader does not substitute for the usual splice leader trans-splicing, since the 3' cap must be added again, or else the transcript will not be translated. That now-nuclear gene-attached splice leader ends up being completely useless, and is able to gradually degrade into benign junk, provided it doesn't mess with the translation of the gene. What is really cool is that one can actually see this gradual degradation, as shown in Slamovits and Keeling 2008 Current Biol:

Mmmm, actual data! Note how the oldest SL piece closest to the gene (on the right) is the most degraded. (Slamovits & Keeling 2008 Curr Biol)

Once the unnecessary splice leader chunk becomes part of the gene, the gene gets transcribed and trans-spliced like any other – meaning it is once again susceptible to replaying that same process of reverse transcription, except this time it already has a relict sequence. It can acquire a second one on top of that. This explains how there can be several concatenated splice leader relics tagging along, like in the above figure.

Splice leader trans-splicing not necessarily promoting reverse transcription – only makes it easier to detect. In other words, it inadvertently makes for a wonderfully convenient system where you can actually track what happens to a gene after it gets reverse transcribed. Once the gene makes its new home, the old gene copy is still present and they generally would be functionally redundant, so the dual-copy state is extremely unstable as ultimately the loss of one of the copies will be tolerated. If the newly transcribed copy is lost, we never see it and thus don't talk about it in the first place. However, once the clean original is lost, only the gene with the crap from the splice leader remains, and reversal to the original state is so improbable it's practically impossible. In other words, this process is a wonderful example of an evolutionary ratchet.

Ratchets are interesting because they confer intrinsic directionality to a system, even in the absense of external pressures (like selection). The accumulation of splice leader junk in the dinoflagellate's genes isn't particularly healthy, nor is it particularly deleterious – it's effectively neutral. However, one can argue that we do have an example of bloated complexity here. Since you can't go back and lose chunks of splice leaders, this ratchet essentially ensures that left to its own devices, this aspect of genome complexity will increase on its own. At a certain point, there will probably be ever-increasing selection against accumulating further splice leaders, and those lineages that go too far will simply die off – the central tendency doesn't care, and the ratchet will keep on going regardless of what selection 'wants'.

This ratchet example is therefore an elegant case of evolutionary direction that's not particularly well explained by the central dogmas of Modern Synthesis or (neo)Darwinism, where selection is the force that crafts order and directionality, with mutation a mere passive provider of material to be molded. I will go into a deeper discussion of this in another post (there's a cool paper coming out soon), but I think it's worth briefly mentioning here too while we're at it. The "mutation" step (to which, I guess, this trans-splicing and reverse-transcription process can be awkwardly attached) here is what provides a drive, a push in a certain direction, and towards increasing complexity, no less (although that last detail is irrelevant). While selection is present and provides constraints (if both genes are lost, for example, the organism dies), it does not do the 'driving' or 'forcing' in this system. Very crudely put, selection here is the passive phenomenon, and mutation is at the wheel.

Another case of intrinsic directionality, but where reversal is allowed, is your garden variety directional bias – where proceeding in one direction is more probable than going backwards. A very basic example of that is if the replication machinery favours a certain type of nucleic acid – left to its own devices, the genome base composition would be skewed in that direction. Boundaries can also induce an apparent directionality, but in this case it's no longer intrinsic... that's, again, a topic for another day.

This idea was a part of the Mutationism theories in the early 20th century, which were a little extreme and perhaps premature, since mutation was far from being even marginally understood at the time. In the usual melodramatic manner characteristic of academia and the scientific community, the pendulum swung far to the opposite extreme, and Modern Synthesis was born. It became heresy to think that mutation itself can actively contribute to direction and order. The field became engulfed in a false dichotomy, where either selection or mutation can actively provide direction, with the modern folk siding with the former. That is a serious mistake and an unnecessary waste of great explanatory potential – you can go so much farther with selection, drift, mutation and recombination all at the wheel, each pulling with different magnitudes in various directions. Well, technically, you wouldn't if you were the thing being pulled – which resonates so well with the absense of 'ascension' or general active directionality in the evolutionary system as a whole. Evolution is a slow, painful, inefficient and rather stochastic process, partly because the cart is being pulled in so many ways.

(The latter part, concerning directional biases and Mutationism, is based on various publications and conversations with Arlin Stoltzfus and Dan McShea, whom I gratefully acknowledge. =D)

References:
McShea, D. (2001). The minor transitions in hierarchical evolution and the question of a directional bias Journal of Evolutionary Biology, 14 (3), 502-518 DOI: 10.1046/j.1420-9101.2001.00283.x

Slamovits, C., & Keeling, P. (2008). Widespread recycling of processed cDNAs in dinoflagellates Current Biology, 18 (13) DOI: 10.1016/j.cub.2008.04.054

Stoltzfus A (2006). Mutationism and the dual causation of evolutionary change. Evolution & development, 8 (3), 304-17 PMID: 16686641

RQ#03 Is there really a non-natural selection?

I haven't done a random question in a while. This is the third one, apparently.

A grocery store still life, primarily Brassica oleracae

Lately I've been involved in some fairly theoretically discussions about evolution, which tend to push one to pay more attention to terminological precision. Or get very confused And get very confused regardless. Additionally, I hang around some biologists with minority opinions on certain aspects of evolution, and ultimately end up talking about evolution differently, to the point of using different words or same words differently. The usual side effects of specialisation. This becomes particularly evident in heated argument with someone outside your tribe – you start speaking slightly different dialects, if you will. Of course, where there's variation, there's opportunity to pick the variant that suits you better. Ideally, that has something to do with accuracy, since we are, hopefully, still attempting to do science and what-not.

Let's start with the easier of the usage and terminology discrepancies – the term 'natural selection'. Is it useful or does the simpler 'selection' make it redundant? I tend to drop the 'natural' part; laziness and word limits may help, but I think there may be valid theoretical or philosophical merit in doing so:

1. 'Natural selection' was initially proposed in contrast to 'artificial selection', which was used as an effective pedagogical/explanatory move. It got the point across, particularly in an age when humans were unquestionably special and distinct from the natural world. Nowadays, few scientists would seriously make a distinction between human and non-human nature in the context of biology, and thus there really is no artificial selection per se. 'Artificial selection' is 'natural selection' performed by humans to pressure their organisms towards traits the humans find favourable. In this case, the humans are part of the environment, playing a similar role to predators, except they breed the variants they like instead of instantly culling them. With no need for an 'artificial selection', is there still a need for 'natural selection', since there no longer is a valid contrast?

2. 'Natural selection' is often equated with adaptation. This isn't to say 'selection' by itself isn't, but 'natural selection' is the variant used most often in popular writing, some of which can be careless and inconsistent with its terminology. While presumably many of the authors do truly understand that selection and adaptation are different things, adaptationism has led some to consider the difference irrelevant. If adaptation is the sole phenomenon responsible for all the observable or cool things in biology, does it really matter if it's used interchangeably with natural selection? When a term is learned and frequently used incorrectly, it is extremely difficult to fix even in an individual, let alone a population. While 'natural selection' is not meant to be conflated with adaptation, it is, and has thus been tainted.

3. Use of 'natural selection' implies that phenomena like sexual selection and kin selection are somehow distinct, or special. These are secondary phenomena, special cases or manifestations of selection. That is, sex and kin selection are subtypes of 'natural selection' and do not lie on equal hierarchical level as it may first seem. While most of the scientific community has no problems understanding this, it is perhaps not the clearest delineation of the terms for the general public or students. This way, we can also keep 'artificial selection' to refer to domestication (although I don't see the necessity in doing so) without it contrasting with the 'natural' kind.

4. This is the least important point, but rather a more personal one. I dislike Darwin-worship; I'm not a 'Darwinian' (nor a "Neo-Darwinian), don't know what that means and frankly don't consider this question relevant now, over a century after Darwin's death. While history of science is indeed fascinating and undeniably worthwhile to learn about, we shouldn't trap ourselves in our history. In fact, I think equating evolution with Darwinism is a bit offensive to all the hard work and frustration of subsequent researchers that have contributed to the field – do they not matter? They work for evolution, not Darwin. 'Natural selection' has been too often tightly associated with 'Darwinism', and often plays a part in Darwin-worship. In other words, the term has acquired some baggage; mind you, not through Darwin but rather through his fervent supporters afterwards.

5. Population geneticists seem perfectly happy with just 'selection'. They're the ones who actually study the mechanisms of this stuff, so if it works for them, perhaps it should be adequate for the rest of us?


I don't mean to nitpick on words and 'mere semantics', but given the difficulty of conveying ideas to those outside your field and the general public, any site of potential confusion is worth trimming if we can. Those on the writing end are also prone to sloppiness and mistakes, so we too are susceptible to the confusion potential. That said, 'natural selection' has stuck around for this long – perhaps there is a beneficial reason I missed out on? This is an honest question – I've never really been formally trained in evolutionary biology save for a basic first year level, and may thus miss large chunks of theory. As I mentioned before, I'm being 'brought up' in some minority circles of evolutionary thought.

Why should we still use 'natural selection'?
Your turn. Just be gentle with the philosophy – I'm rather slow at following complicated abstract theoretical discussions, which is why I do experimental science ;-)

Irremediable Complexity – Science piece

Just wanted to bring to your attention that a new Perspectives piece finally came out in Science this past week, nicely (and in a concise way) summarising Constructive Neutral Evolution – that is, the capacity of non-adaptive and neutral processes to drive a seemingly directed increase in complexity.

Gray MW, Lukeš J, Archibald JM, Keeling PJ & Doolittle WF* 2010 Irremediable Complexity Science 330:920-921

Since non-OA publishers are lame and don't let the unprivileged lower creatures to see their articles, I put up a (hopefully) freely accessible pdf here. Hope the link doesn't die. Read it, it's like my previous ramblings on the subject but more concise and accurate and written by people who know what they're talking about ;-) (I also blogged Ford Doolittle's talk here)

Enjoy!

And yes, part III of my CNE post is coming, as well as the post on Mike Lynch's seminar talk. Let's just say that writing for work does not leave much guilt-free writing juices left for blogging about complicated topics. I mean, if I have the mind and energy to read papers and write about them, I feel guilty not spending that on the work I'm actually paid to do... Should catch up soon enough though, and then the blog monsters shall be unleashed and there will be no respite from the flood of intense protistiness that shall follow, bwahaha!

*The Dal is strong in this list...

Research proposal ramblings – Eukaryotic cellular evolution

Have a research proposal to do in a week for a graduate research fellowship (which also contributes to my spotty blogging as of late). Figured I'd try out Rosie's strategy of blogging to generate ideas and sort things out a bit. Would really appreciate feedback + discussion, particularly criticisms. This is my first time doing one of these, so I'm a total clueless n00b. This isn't my proposal, obviously, but rather a pre-draft of a pre-proposal draft, to lay out some thoughts ;-)

And this stuff needs to be narrowed down A. LOT. I'm well aware of that!

Topic: Eukaryotic cellular evolution

Or "cellular evo-devo"; of protists, obviously ;-)

Possible subtopics:

- The role of non-genomic ('cellular') inheritance in the broader context of evolution

- The relative extent of cellular inheritance in various unicellular and multicellular examples, in relation to size/"complexity", effective population size, mutation rates, whatever. (no idea how I'd do that yet though)

- Tracing the path of individual components in eukaryotic evolution, such as specific protein families, etc. Comparative work.

- ?

Background

Early evolutionary biology focused on the macroscopic level, such as general morphology of an organism and its behaviours in relation to its ecology. Later developments in molecular techniques have largely shifted the focus of the field towards the molecular scale, focusing on genes and proteins. In doing so, the organisms have been reduced to mere genomes – their cellular and developmental contexts largely forgotten. While evo-devo aims to revive the developmental aspect of evolution in multicellular organisms, little has been done to approach evolutionary biology on the level of individual cells, particularly in unicellular lifeforms. Cellular biology is crucial to a properly holistic understanding of evolution, and since the vast majority of organisms on earth, both in quantity and in diversity, are cells, time is ripe to investigate the role of extra-genomic cellular hereditary processes and their role in organismal evolution as a whole.

The first requirement of studying cellular evolution is good sampling. This demands a good phylogeny, and well-understood cell biology among non-Animal/Fungal/Plant organisms. Phylogeny together with cell biology must then be used in attempt to reconstruct ancestral states in order to seek out potential patterns and correlations. These patterns must arise several times independently, in order to have a decent independent sample size. However, at this point it's still only comparative biology; to make it to the level of evolutionary theory, predictive models must be inferred and tested from these patterns – this would ultimately require tying it in with the entire range of biological topics, from biochemistry to population genetics and ecology.

Of the above steps, only the phylogeny has begun to more-or-less solidify, at least enough to begin doing comparative work, if enough well-developed model systems exist in the attention-starved areas of the tree. However, the organismal & cell biology side does not fare as well: of the non-Animal/Fungal/Plant eukaryotes, only the medically important intracellular parasites like Plasmodium, Giardia and Trypanosomes have been well-studied (in the molecular cell biology sense), as well as cellular slime mould Dictyostelium due to its assumed significance in the evolution of multicellularity. To a lesser degree, ciliates Tetrahymena and Paramecium have been studied, as well as diatoms and oomycetes. This leaves huge swaths of phyla severely underrepresented, including practically all of Rhizaria. It must first be established whether anything is salvageable from what has been done to date.

(It would be unwise to rely on developing a novel model system for a PhD project, as potentially awesome as some candidate species may be. Maybe on the side, somehow (I really want Allogromia!))

As an aside, the close relatives of a potential model system should be examined to evaluate how well this model represents the group – picking at outrageously derived system would not be preferred for comparative work, although it would be quite biologically informative in its own right. Some of model candidates may have annoying quirks for certain types of things – eg. Paramecium undergoes autogamy every couple of weeks and destroys its somatic nucleus, thereby being ill-suited for molecular work.

To summarise, studying cellular evolution would require further development of protistan model systems, as well as extensive comparative work between them. Project could focus either on using what's already there for widescale comparative and theoretical work, or picking a single system and working on it specifically.

Relevant examples

- directed assembly of ciliate cytoskeletal elements (Grimes & Aufderheide 1991; Sonneborn & Beisson 1965 PNAS; Frankel 1989) A substantial body of work exists on the topic of ciliate development and how a chunk of the cytoskeletal organisation and morphogenesis seem to depend on non-genomic factors; eg the vertical transmission of a surgically inverted row of cilia independently of genomic inheritance. Furthermore, during encystation, hypotrich ciliates lose all basal bodies, and morphogenesis must happen anew. Some altered traits are lost after encystation, some are not. A mysterious 'organising centre' seems to exist in Oxytricha that determines defining features of the new morphology.

- endosymbiotic bacteria (eg. Görtz 2006 The Prokaryotes) of ciliates as a model for cytoplasmic inheritance of more tangible/quantifiable things.

- organellar inheritance, but that's been done to death already. Relative to above two cases, I mean! (before they kick me out of the lab for saying that...)

Potential projects

A lone tumbleweed rolls across the vast expanses of the chilling mind desert as the ruins of a derelict ghost town stand as a ghastly reminder of the Mind's complete and total absense. A slanted cracked wooden door of the saloon creaks softly in the winds of confusion, seemingly bemoaning the long-gone days of vibrant endless drinking...OH, THERE, I GOT IT! I need beer, the cause of and solution to all research problems!

- Continue the ciliate morphogenesis work of the 60's-80's. Look for general evolutionary principles, if feasible. [something specific goes here]

- Cytoplasmic inheritance of endosymbiotic bacteria and their genomes; effects of various things on that; comparing patterns between different [independent] clades? [something specific and intelligent goes here]

- mapping known cell biological traits onto modern eukaryotic phylogeny, look for patterns. Protein trees, for starters. Eg, Jékély and Cavalier-Smith type of work.

- ?

- lock self in a dark closet microscopy 'room', grab Allogromia or Stentor or Oxytricha or something, work on its molecular cellular biology, screw the whole broad research questions thing. Play with new protists on the side. Be a real hardcore protistologist. Shun everyone else. Damn, so bloody tempting...

Considering the cytoskeleton is the best part of the cell, and tubulin kicks actin's sorry little ass, my hands are seriously itching to do some in vivo fluoresent labelling on various cytoskeletal components in foram reticulopodia, and...well, that's a career right there. Especially when their microtubules grow 10x faster than those of any other eukaryote. (not sure how foram genomes go – could be a pain to work with)

But ciliates are cool too. And Warnowiid dinoflagellates with their awesome 'camera eyes', and radiolarians, and all these other things that aren't even culturable yet. Damn.

Broader impacts, justification

...heh. Should probably have something vaguely resembling a faint outline of a potential project before even considering these. Content first, embellishments after, even if the latter can appear to be more important at times.

---

Problem with cellular evolution at the moment is that we don't even have a decent grasp of cell biology yet, even in multicellular model divas like Arabidopsis and C.elegans. Not even getting into the phylogenetic sampling issues and lack of extant theoretical framework.

And we haven't even mentioned the prokaryotic cells yet. Yes, they have cell biology too, not just bags of biochemistry. No, most people haven't realised that yet, and/or don't care. Yes, we're fucked.

The very idea of cellular evolution almost looks impossible at the moment. So I really want to do it!

Ok, now sink your teeth in and demolish my very dream of making it in my foolish career choice. Start discussion. I need ideas. My mind is full of arcane protist taxa to think straight anymore (did you know Pseudospora was reported in 1905 to produce uniflagellate gametes? Did you care? Lookie, I'm soooo employable! Transferrable skills galore!)

Diagrams that make me cry, part LargeNumber

I was calmly blogging about real content, of course procrastinating simultaneously by writing pieces of stuff for work, as well as planning on possibly contemplating actually doing the readings for tomorrow's paleontology class. In the intro chapter titled "Fossils and Evolution", we get shown the following figure as The Taxonomic System, with a mention in passing the there have been some minor adjustments in the past, such as Three Domains, etc. Of course, such trivial taxonomic nitpicking is apparently of no use to paleontology students, so we'll use the horribly outdated Whittaker 1963 classification which should never be seen outside history of science lectures...

Aaaaaaaaaaah! Gotta love the 3:2 vertebrate:invert ratio too. Apparently bryophytes and 'pteridophytes' don't actually exist either. My own kingdom makes me weep. Also, for personal sanity, ignore the "Protoctista". That's just Margulis trying to feel special. And somehow succeeding outside her own field. (Levin 1999 Ancient Invertebrates and Their Living Relatives. Prentice Hall, NJ)

'Oh but the textbook's from 1999, cut 'em some slack with the volatile ever-changing taxonomy mess!' If they had used the Woese tree, which I have other personal issues with, I'd be fine with it. Of course, Woese tree in 2010 is a bit irritating, but I can still live with that. But...what truly adds insult to the injury, and rubs various salts even deeper into the bleeding wounds of my inner soul – THAT FUCKING PIE CHART!

"Proportions of members of each kingdom living today"

Hang on, gonna go break some furniture with energetic *headdesk* maneuvers. And my forehead with epic *facepalming*. BRB.

...ok, back. Lab benches are quite sturdy, it turns out. I'll just let Star Trek and internet memes take care of this:

Source: The Internets. All of them.

And they teach this as an uncontroversial, neutral fact. When, in fact, reality looks more like this:

Proportions of total marine biomass and abundance occupied by the only noticeable taxonomic groupings. Also, anyone who replaces "eukaryotes" with "protists" must be pretty freaking awesome. And/or actually understand biodiversity. (Suttle 2007 Nature Rev Microbiol)

I don't mean to bash the textbook itself. Aside from the little taxonomical issue there, the rest of the book seems quite interesting and perhaps a great source for paleontology (not being of that field, I have little idea). It just bothers me when something so cheap and simple to fix is left ignored and perpetrated on and on as students fail to learn any better, and teach what the learned, and further students learn that, and so on, ad infinitum, until we're left wondering why modern anthropology graduates seem to think evolutionary biology progressed little since Darwin's time. It's kind of annoying. And detrimental to efficient progress in all relevant fields. Not even asking for a new section in the book; just fix what's already there!


Ok, my blogging spirit is back now. Time to write up actual posts, relying on the creative writing juices (if any) unlocked by the power of RANT.

Clickable Tree of Eukaryotes (Katz Lab)

For a while I've been contemplating on considering to con someone into making a clickable tree for me, allowing one to zoom in and click genus names leading to further info/pictures/whatever. Of course, I'd be far too lazy to actually execute such a project, especially given my lack of programming skills, and lack of faith in the stability of current phylogenies... luckily, I recently discovered some nice people already took care of that, and produced a really awesome tree:

The genus names lead to their respective Micro*scope pages (with pictures)! (Parfrey and Katz, http://www.science.smith.edu/departments/Biology/lkatz/EuTree2009/Eutree09.html; relevant literature: Parfrey et al. 2006 PLoS Genet, 2010 Syst Biol)

This is the eukaryotic tree of life sensu Katz Lab. Being on the opposite side of the continent, the people here have some differing opinions on the subject (my diagram – seriously due for an update – kind of reflects local influences). As you may have noticed from the bounty of polytomies (multiple branches at a single node indicating uncertainty in branching order), the Parfrey and Katz tree is quite conservative, which is probably a good thing. For pedagogical purposes, however, I still think it's better to go ahead with the supergroups, while mentioning the frailty of some, as it helps organise the organisms and dispells the common notion of Protista being just an amorphous grab-bag of microbial crap that doesn't fit. They run the show, it is WE who 'don't fit'...

For research purposes, one must strive to keep track of the certainty of each and every piece of data or hypothesis one works with. Of course, that's overwhelming to n00bs people outside the field, so the shakiness of some models tends to be glossed over. Also, most people don't care.

Speaking of things normal people don't care about, I was quite shocked by the disappearance of Archaeplastida as a clade -- the locals give off the impression Archaeplastida is among the healthier of the supergroups. Excavates, on the other hand, are acknowledged to be somewhat 'meh' as a clade by some of the people working on them. Hacrobia is rumoured to be practically dead anyway, so I'm just keeping that label for the sake of categorising things that may at best turn out to be paraphyletic (which I'm ok with informally), or at worst, grotesquely polyphyletic in ways that would make Heliozoa and Rhizopodia cry. Also, the Stramenopiles are sister to Rhizaria as opposed to Alveolata ("our" order goes (Rhiz,(Stram,Alv))). I find that weird. Although, on the second though, why the hell not. But local folklore has it that Stram+Alv are a pretty solid grouping. Then again, local folklore sings praises to the Chromalveolate Hypothesis... As an innocent, defenseless cell biologist, I'll just hide in the corner until this blows over...

Also, note that the tattered remnants of the 'supergroups' themselves are horribly politomised. Recall how the animal phylogeny tends to have a comb-like branch structure along the 'base' -- ie, among the earlier divergence events, only one group went on to diversify in ways we notice. Then, shortly before the Cambrian diversification event ('explosion' my ass), a bunch of divergences happened that later did lead to multiple lineages that became diverse, in ways we notice. But prior to that, it seems that animal evolution proceeded at a fairly "gradual" pace, according to some anyway. In terms of extant descendants anyway. But in any case, there are ample opportunities for an illiterate journalist (or scientist) to commit the "primitive animal" fallacy.

This error comes much more difficult in the eukaryotic evolution scenario, that is, if only those illiterates knew a thing or two about the modern phylogenies. This is because apparently, very few early-branching 'undiversified' taxa exist, if none at all. Hard to explain without a tree to show, but it seems like the major eukaryotic supergroups rapidly exploded, either soon after the origin of eukaryotes, or all the earlier-diverging clades disappeared without a trace.

This is a question of the 'tempo and mode' of evolution -- the rate and extent of diversification. It's a rather fuzzy concept, as it's quite difficult to establish what diversity is and how to measure it. Considering we biologists don't even know what a species is (and linguists, I'm told, know not what a word (or language), is...), comparing diversity is very difficult. There are some vague tendencies, but that's all they are. Or so it seems anyway -- perhaps I missed something. I guess it's hard to compare the extent of diversity when you reject ranked taxonomy. Zoologists, at least in the past, have used phyla as an indicator, which were somewhat based on the body plan. Whether it's a valid indicator is a whole other topic, but we lack such luxuries in the microbial realm anyway. This topic deserves a proper post someday...

What I was trying to get at, before almost drowning in caveats and disclaimers there, is that the major clades of eukaryotes have arisen rapidly and seem to have left no residual 'basal'/'stem' taxa, making it very difficult to resolve the relationships between them. Resolving recent 'explosions' is quite doable, as is resolving more gradual evolution in the distant past...rapid explosions in the distant past are one hell of a bitch to deal with, which is why much of the deep phylogeny remains a mystery.

How I managed to go off on this tangent eludes me. I see trees, I start chatting about them, ain't nothin' I can do 'bout that.

It being the start of the school year accompanied by an ominous influx of undergrad cooties *shudder*, I'm going to be on slow blogging mode for another week or so. So use that tree to entertain yourselves -- in fact, this tree and ToLweb make my blogging kind of redundant =P (shhh...) Fear not, since I still need to feel useful from time to time, my protists shall keep on coming.

Relevant papers to the Parfrey & Katz tree: (should be accessible)
Parfrey, L., Barbero, E., Lasser, E., Dunthorn, M., Bhattacharya, D., Patterson, D., & Katz, L. (2006). Evaluating Support for the Current Classification of Eukaryotic Diversity PLoS Genetics, 2 (12) DOI: 10.1371/journal.pgen.0020220

Parfrey, L., Grant, J., Tekle, Y., Lasek-Nesselquist, E., Morrison, H., Sogin, M., Patterson, D., & Katz, L. (2010). Broadly Sampled Multigene Analyses Yield a Well-Resolved Eukaryotic Tree of Life Systematic Biology DOI: 10.1093/sysbio/syq037

Criminally photosynthetic: Myrionecta, Dinophysis and stolen plastids

The microbial world is full of vicious beasts. Yes, much of microbial life is cute and cuddly in one way or another. But that doesn't stop many of them from making wolverines seem docile by comparison. There is an entire mafia out there built around...organ theft; including some multicellular players as well, in case you thought animals were saintly. Today we'll look at some famous thieving masterminds of the plastid black market, but keep in mind that there are many more fascinating relationships involving keeping entire organisms or their parts alive within the host, and vastly more oddities that have still escaped human attention (not hard to do, actually).

Let's start off the messy subject with a pretty diagram summarising the major plastid hoarding events of the [moderately] distant past:

Pac-Man!* Today all we need to do is appreciate the overall big picture: there were numerous symbiotic events and by about tertiary endosymbiosis, it gets messy. Not pictured are the cases of more-or-less transient kleptoplasty (plastid-theft), which would do serious harm to the readability and aesthetic qualities of this diagram. (Keeling 2004 Am J Bot; free access) For those keen on extra gory details of plastid endosymbiosis, see this recent review.
*If somebody were to make a game of Pac-Man: Endosymbiosis Edition...

Today's plastidial saga will involve an arduous journey from the cyanobacterium to the red algal endosymbiont of the cryptomonad, to the subsequent ingestion by a ciliate and a dinoflagellate. In fact, just keep in mind that the cryptomonad itself is the result of a hungry heterotroph getting a habit of devouring red algae and developing a case of terminal indigestion, ultimately gaining a plastid and plastid-targetting genes in its own nucleus. The cryptomonad in particular happens to be really awesome in another way: it actually still retains the original, eukaryotic, red algal nucleus of its former prey! That nucleus has been badly shrunk in the wash, and the genome is essentially on crack, but that's a long story for some other day.

Just so you get an idea of what a cryptomonad roughly looks like:

Cryptomonas. Note its very diminutive size. Source: Micro*scope.

We're about to move on to the sleazy thieving ciliates and dinoflagellates. But first, we must establish how kleptoplasty (lit. plastid theft) differs from endosymbiosis. To clarify, I use 'symbiosis' as a general term for an intimate interaction between two different species, including parasitism, mutualism and commensalism. Thus, an endosymbiont needn't feel the same way about the relationship as its host, and very often doesn't. Keep in mind that it is often not very obvious which exact category the symbiosis falls into, as nature doesn't particularly care for our naming fetish.

Endosymbiosis, in the context of organelles and other intracellular stuff, typically entails the complete engulfment of another organism by the cell. Once gene transfer occurs between the genomes of the two organisms, some declare the endosymbiont is now officially an organelle. The endosymbiont-organelle debate is old, stale and utterly pointless; thus, as I have declared in a previous post, I like to call plastids and mitochondria 'endosymbionts' and the more questionable cases, like Perkinsela, 'organelles'. That way, I can piss off just about everyone. Ha!

Then there is the much-awaited plastid theft, where only the plastid itself of the failed endosymbiont is retained, with the rest of it typically digested away. The katablepharid Hatena which Labrat wrote a wonderful post about (as well as Merry at Small Things Considered), is a striking case of kleptoplasty (and only discovered this past decade!). The intensity of kleptoplasty, as well as endosymbiosis, vary greatly from transient plastids (or endosymbionts) that are not essential to the host, to mostly permanent plastids or endosymbionts that are retained indefinitely, capable of reproducing on their own, and completely obligatory for the host's survival. This is nicely summarised in this diagram from a recent review on acquired photosynthesis by Stoeker et al 2009:

Two ways to get a plastid: 1) steal a plastid-bearing alga and lock it in your basement keep it alive within you (endosymbiosis); 2) mug the alga, steal its plastid and try to keep it alive yourself. Along the two paths lie multitudes of intermediate steps different in the persistence of the plastid (how long it lasts) and how dependent the host is upon it. (Stoecker et al. 2009 Aquat Microbiol Ecol)

In the endosymbiotic pathway, nucleomorphs (and the original plastidial prokaryotic genome) suggest the permanent associations we know among the 'normal' algae come from the endosymbiotic path, as there is evidence for whole host retention at some point. However, the data do not entirely rule out some independent secondary plastid acquisition via kleptoplasty rather than endosymbiosis. As for tertiary plastidial symbionts, it gets fun. The classic persistent cases like Kryptoperidinium tend to have a whole endosymbiont, nucleus and all, so the endosymbiotic pathway is also more likely, cut things like Dinophysis, on the other hand, are just weird.

Now, at last, our long-awaited thief: the ciliate Myrionecta rubra (=Mesodinium rubrum):

Myrionecta rubra (originally Mesodinium rubrum); c - cirri; ChC - chloroplast complexes; ECB - equatorial ciliary band (Taylor et al. 1969 Nature) Right: SEM of Myrionecta by Takayama Haruyoshi (more awesome micrographs here)

As you can see, this ciliate bears plastids - a rather non-ciliate activity. In fact, if you slice it up, you'll find that the plastids are very carefully arranged at the periphery:

N - cryptomonad nucleus; M - ciliate macronucleus (note the difference in chromatin organisation); note how the plastids are not only predominantly on the cell periphery but also tend to all face outward! (Oakley & Taylor 1978 Biosyst)

The ciliate captures a cryptophyte, takes its plastids -- along with the nucleomorphs, pyrenoids and other plastid-associated stuff, as well as cryptomonad mitochondria -- and packages them up in their own little compartments. Furthermore, the nucleus is also retained and consistently packaged in an entirely separate package from the plastids. Quite remarkably, the cryptomonad nucleus remains transcriptionally active! (Apparently, Elio beat me to it in 2007. Grrr) Presumably, maintaining an active host nucleus would help keep the plastids functional longer.

Oddly enough, I have difficulties finding anything on the exact process of crypto acquisition - I initially thought it just phagocytoses them, but a friend of mine studying weird plastid aquisition thinks they may actually employ myzocytosis - sucking out the contents of its prey through a 'straw', like many other alveolates do: this may explain the segregation and separate enveloping of the plastid and crypto nucleus. This would require Myrianecta to be quite fast and well-coordinated; the speed is there as it tends to jump instead of moving gradually (details here).


There is a plot twist to this story. A stroke of irony, or poetic justice, or karma if you're into such things. The thieving ciliate itself gets mugged...by a dinoflagellate!

At first glance, Dinophysis caudata is a normal photosynthetic dino, which isn't particularly surprising as roughly half of them are (most with their own plastids). Dinophyceans are quite trippy morphologically, which made it even more frustrating that Dinophysis appeared impossible to culture, despite being photosynthetic. For a while, no one could figure out what exactly was wrong with it. Turns out, its plastids aren't its own, and are rather cryptomonad-like. Great, so it kleptoplasties the cryptos, let's just grow it in a jar full of them! Again, no luck - for some reason, Dinophysis appeared incapable of ingesting the cryptomonads!

It was all rather perplexing until someone figured out the problem in the 2000's, publishing the first successful culturing attempt in 2006 (Park et al. 2006 Aquat Microbiol Ecol). Here's what was missing:

Dinophysis (the jug-like thing with a conspicuous flagellum) sucking the plastids out of Myrionecta, who's rolled up into a small, whimpering ball by this point. (Park et al. 2006 Aquat Microbiol Ecol)

Not only is Dinophysis caudata a stinkin' thief, but it can't even do the primary stealing itself - the dino requires Myrionecta to do all the dirty work of packaging up the plastids. But it gets messier. First, a summary of the plastid's plight:

Dinophysis ingests plastids from the ciliate Myrionecta, who in turn stole them from a cryptomonad. Who, if you recall, obtained it a long time ago as a red algal endosymbiont. Who, of course, obtained the original plastid as a cyanobacterial symbiont. I think it ends there though. That poor cyanobacterial genome has been through a lot! (Wisecaver & Hackett 2010 BMC Genomics)

Now, whether Dinophysis also bears proper plastids of its own is up to heated debate at the moment. It looks like I'm not the only one thoroughly confused by it, and sorting out this issues is slightly beyond the responsibilities of a mere blogger at the moment, so let's leave this part of the story explicitly vague. It seems like Dinophysis may somehow supplement its own stock with the stolen plastids, as it appears to have plastid-targetting genes in its own genome (Wisecaver & Hackett 2010 BMC Genomics). However, there are also cases of Dinophysis carrying plastids that appeared very non-cryptomonad, and most likely to be of dinoflagellate origin (Garcia-Cuetos et al. 2009 Harmful Algae).

The chaos is quite understandable: it is actually very difficult to determine the nature of a relationship between two organisms, especially on the microscopic scale, and especially when one is inside another. It's often hard to distinguish a permanent from a transient relationship, and a mutualistic from a parasitic one. While there is strong direct evidence that the dino sucks plastids out of Myrionecta, that does not necessarily mean all of its plastids originated there. Or that it lacks its own (though that would make sense). Or more importantly, that the various research teams are even looking at the same bloody organism! Speaking of which, Myrionecta and Dinophysis appear to be in a 'bit' of taxonomic mess too, so I'll just let the professionals fight it out amongst themselves.

While that's going on, one cannot help but wonder how many such 'unconventional' relationships there really are. Food webs are not as direct as people think, the once one peers a little further than the usual stereotyped interactions (predator, parasite, prey, producer, whatever), ecology actually becomes an interesting (admittedly, fascinating!) subject. On that note, I think we should really be careful when trying to force terrestrial and macroscopic ecological terms onto the microbial world -- and by careful, I think we should perhaps come up with a system specialised for microbial life from the very beginning. While we seldom see one animal rip out an organ of another and keep it alive for itself, organelle theft is actually not all that uncommon. Life on the cellular level is weird to us, and many traditional terms simply fail to describe it.

There's a whole black market of utterly bizarre microbial interactions out there. We are only scratching the surface.

References
Garcia-Cuetos, L., Moestrup, �., Hansen, P., & Daugbjerg, N. (2010). The toxic dinoflagellate Dinophysis acuminata harbors permanent chloroplasts of cryptomonad origin, not kleptochloroplasts Harmful Algae, 9 (1), 25-38 DOI: 10.1016/j.hal.2009.07.002

Johnson, M. (2010). The acquisition of phototrophy: adaptive strategies of hosting endosymbionts and organelles Photosynthesis Research DOI: 10.1007/s11120-010-9546-8

Johnson, M., Oldach, D., Delwiche, C., & Stoecker, D. (2007). Retention of transcriptionally active cryptophyte nuclei by the ciliate Myrionecta rubra Nature, 445 (7126), 426-428 DOI: 10.1038/nature05496

Keeling, P. (2004). Diversity and evolutionary history of plastids and their hosts American Journal of Botany, 91 (10), 1481-1493 DOI: 10.3732/ajb.91.10.1481

OAKLEY, B., & TAYLOR, F. (1978). Evidence for a new type of endosymbiotic organization in a population of the ciliate Mesodinium rubrum from British Columbia Biosystems, 10 (4), 361-369 DOI: 10.1016/0303-2647(78)90019-9

Park, M., Kim, S., Kim, H., Myung, G., Kang, Y., & Yih, W. (2006). First successful culture of the marine dinoflagellate Dinophysis acuminata Aquatic Microbial Ecology, 45, 101-106 DOI: 10.3354/ame045101

Stoecker, D., Johnson, M., deVargas, C., & Not, F. (2009). Acquired phototrophy in aquatic protists Aquatic Microbial Ecology, 57, 279-310 DOI: 10.3354/ame01340

TAYLOR, F., BLACKBOURN, D., & BLACKBOURN, J. (1969). Ultrastructure of the Chloroplasts and Associated Structures within the Marine Ciliate Mesodinium rubrum (Lohmann) Nature, 224 (5221), 819-821 DOI: 10.1038/224819a0

Wisecaver, J., & Hackett, J. (2010). Transcriptome analysis reveals nuclear-encoded proteins for the maintenance of temporary plastids in the dinoflagellate Dinophysis acuminata BMC Genomics, 11 (1) DOI: 10.1186/1471-2164-11-366